The invention relates to chemotherapy and drug resistance.
Cancer chemotherapy commonly involves the administration of one or more cytotoxic or cytostatic drugs to a patient. The goal of chemotherapy is to eradicate a substantially clonal population (tumor) of transformed cells from the body of the individual, or to suppress or to attenuate growth of the tumor. Tumors may occur in solid or liquid form, the latter comprising a cell suspension in blood or other body fluid. A secondary goal of chemotherapy is stabilization (clinical management) of the afflicted individual""s health status. Although the tumor may initially respond to chemotherapy, in many instances the initial chemotherapeutic treatment regimen becomes less effective or ceases to impede tumor growth. The selection pressure induced by chemotherapy promotes the development of phenotypic changes that allow tumor cells to resist the cytotoxic effects of a chemotherapeutic drug. Often, exposure to one drug induces resistance to that drug as well as other drugs to which the cells have not been exposed.
Cell cycle checkpoints are regulatory systems that control the order and timing of certain events in the cell cycle. These checkpoints are important for ensuring that cells divide properly. For example, DNA damage leads to activation of a cell cycle checkpoint regulatory system that arrests the cell cycle and activates genes involved in repair of DNA damage. This system prevents progression of the cell cycle until the DNA damage has been repaired.
Chk1, a kinase, is thought to be involved in the DNA damage cell cycle checkpoint. Chk1 is thought to participate in the phosphorylation of Cdc25 in response to DNA damage. Phosphorylation of Cdc25 prevents activation of the Cdc2-cyclin B complex thereby blocking mitotic entry.
The present invention concerns checkpoint kinase 1 (Chk1; Genbank Accession No. AF016582; Sanchez et al. (1997) Science 277:1497). Applicants have found that expression of Chk1 is up regulated in certain vinblastin resistant cancer cell lines and in certain adromycin resistant cancer cell lines. Applicants have also found that a ribozyme designed to decrease Chk1 expression can increase drug sensitivity.
Chk1 nucleic acids and polypeptides are useful in diagnostic methods related to identification of drug resistant cells (e.g., cancer cells). Chk1 nucleic acids and polypeptides are also useful in screening methods directed to the identification of compounds that can modulated (increase or decrease) the drug resistance of a particular cell type or multiple cell types.
The invention includes a method for detecting the presence of a Chk1 polypeptide in a sample, e.g., a biological sample. This method features the steps of contacting the sample with a compound which selectively binds to the polypeptide and then determining whether the compound binds to a polypeptide in the sample. In some cases, the compound which binds to the polypeptide is an antibody.
The invention also features methods for detecting the presence of a Chk1 nucleic acid molecule in a sample. This method includes the steps of contacting the sample with a nucleic acid probe or primer which selectively hybridizes to a Chk1 nucleic acid molecule (e.g., an mRNA encoding Chk1); and then determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample.
Also within the invention are kits that include a compound which selectively binds to a Chk1 polypeptide or nucleic acid and instructions for use. Such kits can be used to determine whether cells within a biological sample, e.g., a sample of patient cells, are drug resistant.
The invention features methods for identifying a compound which binds to a Chk1 polypeptide. These methods include the steps of contacting a Chk1 polypeptide with a test compound and then determining whether the polypeptide binds to the test compound. In various embodiments of these methods, the binding of the test compound to the Chk1 polypeptide is detected using an assay which measures binding of the test compound to the polypeptide or using a competition binding assay.
The invention also includes a method for modulating the activity of a Chk1 polypeptide. This method includes the steps of contacting the polypeptide or a cell expressing the polypeptide with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.
In another aspect, the invention provides a method for identifying a compound that modulates the activity of a Chk1 polypeptide (e.g., a Chk1 protein). In general, such methods entail measuring a biological activity of the polypeptide in the presence and absence of a test compound and identifying those compounds which alter the activity of the polypeptide (e.g., alter the ability of Chk1 to phosphorylated Cdc25). One such method includes the steps of contacting the polypeptide with a test compound and then determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.
The invention also features methods for identifying a compound which modulates the expression of a Chk1 nucleic acid or a Chk1 polypeptide by measuring the expression of the nucleic acid or polypeptide in the presence and absence of a compound.
Other aspects of the invention are methods and compositions relating to drug resistance. A xe2x80x9cdrug-resistant phenotypexe2x80x9d refers to a cellular phenotype which is associated with increased survival (compared to a less drug-resistant cell) after exposure to a particular dose of a drug, e.g., a chemotherapeutic drug, compared to a cell that does not have this phenotype. A xe2x80x9cdrug-resistant cellxe2x80x9d refers to a cell that exhibits this phenotype. Drug resistance can be characterized by lower intracellular concentration of a drug compared to a non-resistant cell or a less resistant cell as well as altered ability of a drug to affect its target compared to a non-resistant cell or a less resistant cell. Drug resistance is described in detail by Hochhauser and Harris ((1991) Brit. Med. Bull. 47:178-96); Simon and Schindler ((1994) Proc. Nat""l Acad Sci USA 91: 3497-504); and Harris and Hochhauser ((1992) Acta Oncologica 31:205-213); Scotto et al. ((1986) Science 232: 751-55). Multi-drug resistance can be associated with, for example, altered composition of plasma membrane phospholipids; increased drug binding and intracellular accumulation; altered expression or activity of plasma membrane or endomembrane channels, transporters or translocators; altered rates of endocytosis and associated alteration in targeting of endosomes; altered exocytosis; altered intracellular ionic environments; altered expression or activity of proteins involved in drug detoxification; and altered expression or activity of proteins involved in DNA repair or replication.
Also within the invention is a method of determining whether a cell has a drug-resistant phenotype by measuring the expression (or activity) of Chk1 in the cell and comparing this expression to that in a control cell. Increased expression (or activity) of Chk1 in the cell compared to the control cell indicates that the cell has a drug-resistant phenotype. In one embodiment of this method, Chk1 expression is determined by measuring Chk1 protein (e.g., measuring Chk1 protein using an antibody directed against Chk1). In another embodiment, Chk1 expression is measured by quantifying mRNA encoding Chk1 or the copy number of the Chk1 gene. In another embodiment Chk1 activity is measured using any assay which can quantify a biological activity of Chk1.
The invention also includes a method for modulating the drug resistance of a cell by modulating Chk1 expression or activity within the cell. Thus, in one embodiment, the drug-resistance of a cell is reduced by contacting the cell with a molecule (e.g., an antisense nucleic acid molecule) that reduces the expression of Chk1 within the cell.
Another aspect of the present invention is a method of improving effectiveness of chemotherapy for a mammal having a disorder associated with the presence of drug-resistant neoplastic cells. In this method, a chemotherapeutic drug and a molecule that reduces expression of Chk1 can be co-administered to a mammal. Alternatively, the chemotherapeutic drug can be administered before or after administration of the compound that reduces expression of Chk1.
The invention also includes a method of identifying a compound that modulates the drug resistance of a cell by first contacting the cell with a test compound and then measuring and comparing Chk1 expression in the cell exposed to the compound to Chk1 expression in a control cell not exposed to the compound. The compound is identified as modulator of drug resistance when the level of Chk1 expression in the cell exposed to the compound differs from the level of Chk1 expression in cells not exposed to the compound. In one embodiment of this method, the cell has a drug-resistant phenotype. In another embodiment, the cell is a mammalian cell. This method may also include an optional step of measuring the drug resistance of the cell in the presence of the identified modulator of drug resistance. The Chk1 modulating compounds that are identified in the foregoing methods are also included within the invention.
The invention also features a method of treating a mammal suspected of having a disorder associated with the presence of drug-resistant cells. This method includes the steps of determining whether a mammal has a disorder associated with the presence of drug-resistant cells having increased Chk1 expression (e.g., drug-resistant cancer), and administering to the mammal a compound that sufficiently reduces the expression of Chk1 so that the drug resistance of the cells associated with the disorder is modulated (i.e., reduced).
Another feature of the invention is a method for treating a patient having a neoplastic disorder (e.g., cancer) by administering to the patient a therapeutically effective amount of a compound that decreases the expression of Chk1.
In the context of cancer treatment, the expression level of Chk1 may be used to: 1) determine if a cancer can be treated by an agent or combination of agents; 2) determine if a cancer is responding to treatment with an agent or combination of agents; 3) select an appropriate agent or combination of agents for treating a cancer; 4) monitor the effectiveness of an ongoing treatment; and 5) identify new cancer treatments (either single agent or combination of agents). In particular, Chk1 may be used as a marker (surrogate and/or direct) to determine appropriate therapy, to monitor clinical therapy and human trials of a drug being tested for efficacy and in developing new agents and therapeutic combinations.
Accordingly, the present invention provides methods for determining whether an agent, e.g., a chemotherapeutic agent such as vinblastin, will be effective in reducing the growth rate of cancer cells comprising the steps of: a) obtaining a sample of cancer cells; b) determining the level of expression in the cancer cells of Chk1; and c) identifying that an agent will be effective when Chk1 is not expressed or is expressed at relatively low level. Alternatively, in step (c), an agent can be identified as being relatively ineffective when to use to treat the cancer when Chk1 is expressed or is expressed at relatively high level.
As used herein, an agent is said to reduce the rate of growth of cancer cells when the agent can reduce at least 50%, preferably at least 75%, most preferably at least 95% of the growth of the cancer cells at a given concentration of the agent. Such inhibition can further include a reduction in survivability and an increase in the rate of death of the cancer cells. The amount of agent used for this determination will vary based on the agent selected. Typically, the amount will be a predefined therapeutic amount.
As used herein, an agent is defined broadly as anything that cancer cells can be exposed to in a therapeutic protocol. In the context of the present invention, such agents include, but are not limited to, chemotherapeutic agents, such as anti-metabolic agents, e.g., Ara AC, 5-FU and methotrexate, antimitotic agents, e.g., taxol, vinblastine and vincristine, alkylating agents, e.g., melphanlan, BCNU and nitrogen mustard, Topoisomerase II inhibitors, e.g., VW-26, topotecan and Bleomycin, strand-breaking agents, e.g., doxorubicin and DHAD, cross-linking agents, e.g., cisplatin and CBDCA, radiation and ultraviolet light. A preferred agents is doxorubicin.
The agents tested in the present methods can be a single agent or a combination of agents. For example, the present methods can be used to determine whether a single chemotherapeutic agent, such as methotrexate, can be used to treat a cancer or whether a combination of two or more agents can be used.
Cancer cells include, but are not limited to, carcinomas, such as squamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synoviosarcoma and mesotheliosarcoma; leukemias and lymphomas such as granulocytic leukemia, monocytic leukemia, lymphocytic leukemia, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or Hodgkins disease; and tumors of the nervous system including glioma, meningoma, medulloblastoma, schwannoma or epidymoma.
The source of the cancer cells used in the methods of the invention will be based on how the method of the present invention is being used. For example, if the method is being used to determine whether a patient""s cancer can be treated with an agent, or a combination of agents, then the preferred source of cancer cells will be cancer cells obtained from a cancer biopsy from the patient. Alternatively, cancer cells line of similar type to that being treated can be assayed. For example if breast cancer is being treated, then a breast cancer cell line can be used. If the method is being used to monitor the effectiveness of a therapeutic protocol, then a tissue sample from the patient being treated is the preferred source. If the method is being used to identify new therapeutic agents or combinations, then any cancer cells, e.g., cells of a cancer cell line, can be used.
A skilled artisan can readily select and obtain the appropriate cancer cells that are used in the present method. For cancer cell lines, sources such as The National Cancer Institute, for the NCI-60 cells used in the examples, are preferred. For cancer cells obtained from a patient, standard biopsy methods, such as a needle biopsy, can be employed.
In the methods of the present invention, the level or amount of expression of Chk1 is determined. As used herein, the level or amount of expression refers to the absolute level of expression of an mRNA encoded by the gene or the absolute level of expression of the protein encoded by the gene (i.e., whether or not expression is or is not occurring in the cancer cells).
As an alternative to making determinations based on the absolute expression level of selected genes, determinations may be based on the normalized expression levels. Expression levels are normalized by correcting the absolute expression level of a sensitivity or resistance gene by comparing its expression to the expression of a gene that is not a sensitivity or resistance gene, e.g., a housekeeping genes that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the acting gene. This normalization allows one to compare the expression level in one sample, e.g., a patient sample, to another sample, e.g., a non-cancer sample, or between samples from different sources. Alternatively, the expression level can be provided as a relative expression level. To determine a relative expression level of a gene, the level of expression of the gene is determined for 10 or more samples, preferably 50 or more samples, prior to the determination of the expression level for the sample in question. The mean expression level of each of the gene assayed in the larger number of samples is determined and this is used as a baseline expression level for the gene in question. The expression level of the gene determined for the test sample (absolute level of expression) is then divided by the mean expression value obtained for that gene. This provides a relative expression level and aids in identifying extreme cases of sensitivity or resistance. Preferably, the samples used will be from similar tumors or from non-cancerous cells of the same tissue origin as the tumor in question. The choice of the cell source is dependent on the use of the relative expression level data. For example, using tumors of similar types for obtaining a mean expression score allows for the identification of extreme cases of sensitivity or resistance. Using expression found in normal tissues as a mean expression score aids in validating whether the gene assayed is tumor specific (versus normal cells).
Also within the invention is a method for increasing drug resistance in a cell having an undesirably low level of Chk1 expression by administering a compound that increases the expression of Chk1. Such methods are useful for the protection of non-neoplastic cells during chemotherapy.
The invention features a method for determining whether a test compound modulates the drug resistance of a cell, the method including: a) determining the level of Chk1 expression (e.g., Chk1 encoded by an endogenous or heterologous gene) in a cell in the presence of a test compound; b) determining the level of Chk1 expression in the cell in the absence of the test compound; and c) identifying the compound as a modulator of drug resistance of the cell if the level of expression of Chk1 in the cell in the presence of the test compound differs from the level of expression of Chk1 in the cell in the absence of the test compound.
The invention features a method for determining whether a test compound modulates the drug resistance of a cell, the method including: a) determining the level of Chk1 activity in a cell in the presence of a test compound; b) determining the level of Chk1 activity in the cell in the absence of the test compound; and c) identifying the compound as a modulator of drug resistance of the cell if the level of activity of Chk1 in the cell in the presence of the test compound differs from the level of activity of Chk1 in the cell in the absence of the test compound.
The invention also features a method for determining whether a test compound modulates the drug resistance of a cell, the method including: a) incubating Chk1 protein in the presence of a test compound; b) determining whether the test compound binds to the Chk1 protein; c) selecting a test compound which binds to the Chk1 protein; d) administering the test compound selected in step c) to a non-human mammal having drug resistant cells; e) determining whether the test compound alters the drug resistance of the cells in the non-human mammal; and f) identifying the test compound as a modulator of drug resistance of the cell if the compound alters the drug resistance of the cells in step e).
The invention further features a method for determining whether a test cell has a drug-resistant phenotype, the method including: a) measuring the expression of Chk1 in the test cell; b) comparing the expression of Chk1 measured in step a) to the expression of Chk1 in a control cell not having a drug-resistant phenotype; and c) determining that the test cell has a drug resistant phenotype if the expression of Chk1 in the test cell is greater than the expression of Chk1 in the control cell.
In another aspect the invention features a method of determining whether a test cell has a drug-resistant phenotype, the method including: a) measuring the activity of Chk1 in the test cell; b) comparing the activity of Chk1 measured in step a) to the activity of Chk1 in a control cell not having a drug-resistant phenotype; and c) determining that the test cell has a drug resistant phenotype if the activity of Chk1 in the test cell is greater than the activity of Chk1 in the control cell.
In yet another aspect the invention features a method for determining whether a subject has or is at risk f developing a drug resistant tumor, the method including: a) measuring the expression of Chk1 mRNA in a biological sample obtained from the subject (using, e.g., a nucleic acid molecule that hybridizes to Chk1 mRNA); b) comparing the expression of Chk1 mRNA measured in step a) to the expression of Chk1 mRNA in a biological sample obtained from a control subject not having a drug resistant tumor; and c) determining that the patient has or is at risk of developing a drug resistant tumor if the expression of Chk1 mRNA in the biological sample obtained from the patient is higher than the expression of Chk1 mRNA in the biological sample obtained from the control subject.
In still another aspect the invention features a method for determining whether a subject has or is at risk of developing a drug resistant tumor, the method including: a) measuring the activity of Chk1 in a biological sample obtained from the subject (using, e.g., an agent that binds to Chk1 protein); b) comparing the activity of Chk1 measured in step a) to the activity of Chk1 in a biological sample obtained from a control subject not having a drug resistant tumor; and c) determining that the patient has or is at risk of developing a drug resistant tumor if the activity of Chk1 in the biological sample obtained from the patient is higher than the activity of Chk1 in the biological sample obtained from the control subject.
The invention also features a method for monitoring the effect of an anti-tumor treatment on a patient, the method including: a) measuring the expression of Chk1 in a tumor sample obtained from the patient (using, e.g., a nucleic acid molecule that hybridizes to Chk1 mRNA); b) comparing the expression of Chk1 measured in step a) to the expression of Chk1 in a control sample of cells; and c) determining that the anti-tumor treatment should be discontinued or modified if the expression of Chk1 in the tumor sample is higher than the expression of Chk1 in the control sample of cells.
The invention also features a method for monitoring the effect of an anti-tumor treatment on a patient, the method including: a) measuring the activity of Chk1 in a tumor sample obtained from the patient (using, e.g., an agent that binds to Chk1 protein); b) comparing the activity of Chk1 measured in step a) to the activity of Chk1 in a control sample of cells; and c) determining that the anti-tumor treatment should be discontinued or modified if the activity of Chk1 in the tumor sample is higher than the activity of Chk1 in the control sample of cells.
The invention further features a method for modulating the drug resistance of a cell by modulating Chk1 expression within the cell and a method for reducing the drug resistance of cell by contacting the cell with a molecule which reduces the expression of Chk1 within the cell.
The invention also features a method of increasing the effectiveness of a chemotherapeutic compound in a patient suffering from a disorder associated with the presence of drug-resistant neoplastic cells, the method including: a) administering a chemotherapeutic compound to the patient; and b) administering a compound with reduces Chk1 expression to the patient.
The invention features a method of treating a mammal suspected of having a disorder associated with the presence of drug-resistant cells, the method including administering to the mammal a compound that reduces the expression of Chk1 in the drug-resistant cells, the reduction be sufficient to reduce the drug resistance of the drug resistant cells and a method for increasing the drug resistance of cell that has an undesirably low level of Chk1 expression, the method including exposing the cell to a compound that increases the expression of Chk1.
The invention also features a method for treating a drug resistant tumor in a patient, the method comprising administering to said subject an amount of a Chk1 antagonist effective to reduce drug resistance of said tumor in the patient. In another aspect, the invention features the use of an inhibitor of Chk1 expression, or pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing either entity, for the manufacture of a medicament for the treatment of a drug resistant tumor in a patient.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other features and advantages of the invention will be apparent from the detailed description and from the claims. Although materials and methods similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred materials and methods are described below.